CP for Tumor microarray

Hi!

I am pretty overwhelmed with structuring a pipeline for my fluorescence quantification in my tumor microarray images. For each tumor core(tissue) I have DAPI and a FITC image. I have about 400 images each (DAPI & FITC) and am not sure if I need to overlay my DAPI and FITC images for fluorescence quantification.
I tried to set up a module where I guessed it should look something like Loadimages|correctilluminationcalculate|correctilluminationapply|indetifyprimaryobjects|measureobjectintensity etc. However I have no idea what values to put in ‘module settings’. For example, for identifyprimaryobjects, I’m required to provide input image and values like threshold strategy? Is there anyone who can walk me through it? If anyone with experience from New Orleans can contact me, I’m willing to pay $30/hour as mentoring fee, if not, I’m willing to try an online mentor and we can work something out.

Thanks in advance!

Hi,

It is a little daunting to start image analysis! But glad you asked here.

[quote]…am not sure if I need to overlay my DAPI and FITC images for fluorescence quantification
[/quote]

No, you don’t need to create a color image of your two channels, if that’s what you mean. In fact, it is better to keep them as individual grayscale images.

As for the pipeline help, feel free to upload a couple images here or link via Dropbox, say. Also post your proto-pipeline, so we can take a look.

Just guessing, but for tissue, we usually suggest UnmixColors to start with. The illumination correction modules may not be necessary too, and can cause headaches unless you know you need them. Otherwise, please post your pipeline here. No consulting fee necessary! :smile:

Cheers,
David